This proposal outlines the development of VP22 as a delivery vehicle for protein and DNA molecules to cells in culture. The Phase I proposal demonstrated the power of protein-mediated delivery by expressing and purifying protein fusions with the Antennapedia peptide from E. coli. These fusion proteins were translocated across biological membranes, accumulating in the cytoplasm and nucleus. A new translocating protein, VP22, was recently described and has two significant advantages over the Antennapedia peptide: 1) VP22 can translocate larger peptides and 2) expression of VP22 in a subpopulation of cells results in spread of the protein throughout the entire culture. During the Phase II period, prokaryotic and eukaryotic vectors for the expression and purification of VP22 fusion proteins will be constructed. VP22 fusions will be used to develop systems that can regulate gene expression. VP22 fusions that interact with DNA will be developed and used to investigate the ability of VP22 to deliver oligonucleotides and plasmid DNA to cells in culture. This revolutionary technology has broad applications for the delivery of proteins and DNA to cells that cannot be transfected with current technologies and will provide new techniques to study gene function in all cell types. PROPOSED COMMERCIAL APPLICATIONS: Prokaryotic and eukaryotic vectors will be constructed to allow researchers to express and purify VP22 fusion proteins. VP22 fusions will be constructed to develop an inducible gene expression system and to deliver single chain antibodies to intracellular targets. Reagents based on VP22 will be developed for the delivery of oligonucleotides and plasmid DNA to tissue culture cells that are refractory to current transfection technologies.